Modern biotechnology provides new and/or improved classes of therapeutic and prophylactic agents that treat and prevent diseases, disorders, conditions and infections. Such agents can be made less expensively, more pure and more efficiently.
Protein therapeutic and prophylactic agents have been around for many years. Molecular biology offers the opportunity to find many more of such therapeutics and recombinant DNA technology has made production of proteins a relatively inexpensive undertaking. Similarly, proteins used as vaccine components can be produced in large quantities using such technology. Further, hybridoma technology allows for the production of large quantities of monoclonal antibodies.
DNA and RNA based therapeutic and prophylactic agents are becoming a growing field of product development. Advances in the understanding of biology has led to the development of several fields of endeavor, such as nucleic acid vaccines, antisense compounds and gene therapy, that use DNA- and RNA-based agents.
One problem associated with use of these types of agents is that they can be contaminated with small quantities of molecules whose presence is undesirable. For example, recombinant protein products may be contaminated with plasmid and/or chromosomal DNA. Plasmid DNA usually contains antibiotic resistance genes. The possibility of contamination of protein products by plasmid DNA therefore raises safety and public health concerns which must be addressed. Similarly, nucleic acid-based agents such as plasmid-based vaccines can be contaminated with protein or linear DNA, the administration of either as contaminants being undesirable. Antisense compounds may similarly be contaminated with proteins. The presence of contaminants in nucleic acid-based agents raises safety issues which must be addressed.
In addition to the detection of contaminants in samples of material, modern molecular biology and biochemistry methodologies often include protocols where the detection and quantitation of proteins or nucleic acids is often desirable but where current methods are impractical. For example, the concentration of purified DNA in various buffers or in water is often measured spectrophotometrically at 260 nm using the extinction coefficient of A.sub.260 =1 for a 50 .mu.g/ml solution of double-stranded DNA or for a 40 .mu.g/ml solution of single-stranded DNA. Purified or crude DNA samples can also be measured spectrophotometrically using various assays such as diphenylamine or by fluorescence emitted by the binding of a minor groove binding dye diamidinophenylindole. These methods require several micrograms of DNA and do not distinguish between linear and closed circular DNA. Ethidium bromide staining of agarose gels containing DNA (50-100 ng) is routinely used in a typical molecular biology lab, but estimation of the concentration of stained DNA is complicated by differences in the kinetics of dye binding to various forms of DNA. Southern and dot blot techniques are useful for qualitative analysis, but are imprecise in quantitation of the target molecule due to the large variations in the efficiencies of a multistep procedure.
Of several methods that are currently available for the detection of DNA none is more sensitive than Southern blot/dot blot which allows for detection of nanogram quantities of DNA. However, several limitations render this method useless for the precise quantitative determination of DNA concentrations. Linking the target DNA quantitatively onto a filter, the efficiency of labeling the DNA probe, salt and temperature effects, effects of neutral polymers that favor hybridization, the concentration of probe DNA during hybridization, and finally the stringent washes that remove the unhybridized and some of the hybridized probe, contribute toward the aforementioned lack of precision in Southern and dot blot methodologies designed to quantitate DNA. Current detection/quantification protocols often require samples of a size close to or greater than the size of the specimen of interest. For example, in order to find out how much DNA, RNA or protein is present in a sample, the entire sample may need to be used.
Accordingly, there is a need for methods of detecting and/or quantifying the presence and amount of nucleic acid molecules and proteins. There is a need for methods of detecting and/or quantifying the presence and amount of linear nucleic acid molecules in samples containing circular plasmids. There is a need for methods of detecting and/or quantifying the presence and amount of proteins contaminating samples containing nucleic acid molecules. There is a need for methods of detecting and/or quantifying the presence and amount of nucleic acid molecules contaminating samples containing protein molecules. There is a need for methods of measuring the amount of proteins or nucleic acid molecules present in a sample.